Cobalt chrome-on-cobalt chrome bearing surfaces have been re-introduced despite some concerns regarding potential risks posed by soluble metallic by-products. We have investigated whether there are metal-selective differences between the levels of genetic damage caused to a human cell line when cultured with synovial fluids retrieved from various designs of orthopaedic joint replacement prostheses at the time of revision arthroplasty.

Synovial fluids were retrieved from revision hip and knee arthroplasty patients with bearings made from cobalt chrome-on-cobalt chrome, cobalt chrome-on-polyethylene and stainless steel-on-polyethylene. Control synovial fluids were retrieved from primary arthroplasty cases with osteoarthritis. Synovial fluid was cultured with human primary fibroblasts for 48 hours in a cell culture system under standardised conditions. The “Comet” assay was used with an image analysis system to measure levels of DNA damage caused by the various synovial fluid samples.

Synovial fluids from cobalt chrome-on-cobalt chrome and cobalt chrome-on-polyethylene joint replacements both caused substantial levels of genetic damage as detected by the Comet assay. Synovial fluids retrieved from stainless steel-on-polyethylene joints caused low levels of damage. The difference between these groups was highly statistically significant (p< 0.001). Control synovial fluids from osteoarthritic joints caused minimal changes. Atomic absorption spectroscopy demonstrated that the metal-on-metal synovial fluids contained the highest levels of cobalt and chromium. Different alloys used in orthopaedic implants are associated with different levels of DNA damage to cultured human cells in vitro. We are able to demonstrate that this damage is attributable at least in part to the metal content of the synovial fluid samples. We have no evidence for any long-term health risk to patients with such implants.

Metal-on-metal bearing surfaces have been reintroduced for use in total hip replacement, despite concerns regarding the potential risks posed by metallic by-products. We have compared periprosthetic tissues from metal-on-metal and metal-on-polyethylene hip replacements at revision surgery with control tissues at primary arthroplasty.

Tissues were obtained from 9 control, 25 contemporary metal-on-metal, 9 CoCr-on-polyethylene and 10 titanium-on-polyethylene hip replacement arthroplasties. Each was processed for routine histology with Haematoxylin and Eosin. Quantitative stereological analysis was performed at the light microscopic level.

Metal-on-metal sections showed more surface ulceration and this was correlated with the density of inflammation in the deeper tissues layers. Metal-on-metal tissues displayed a pattern of well-demarcated tissue layers, which were rarely seen in metal-on-polyethylene cases. In metal-on-polyethylene cases, the inflammation was predominantly histiocytic. Metal-on-metal cases by contrast showed a lymphocytic infiltrate with abundant plasma cells. Metal-on-metal tissues showed a striking pattern of peri-vascular inflammation with prominent lymphocytic cuffs especially deep to areas of surface ulceration. Levels of inflammation were higher in cases revised for failure than in those retrieved at autopsy or exploratory surgery. Total replacement and surface replacement designs of metal-on-metal arthroplasty showed similar histological changes. Plasma cells were not seen in any of the metal-on-polyethylene cases. The differences between the patterns of inflammation and cellular infiltration seen in metal-on-metal and metal-on-polyethylene tissues were highly statistically significant.

The pattern and type of inflammation in periprosthetic tissues from metal-on-metal and metal-on-poly-ethylene arthroplasties is very different. Our findings support the conclusion that metal-on-metal articulations are capable of generating a form of immunological response to metallic wear debris that has not been described previously. The incidence and clinical implications of these immunological responses in failed metal-on-metal joints are unknown.

Introduction: Cobalt-chrome particles from metal hip implants can accumulate in the liver, spleen, lymph nodes and bone marrow of patients. This is a concern as studies have reported neoplastic changes in cells of patients with metal implants. The aims of this study were to determine the effect of wear particles generated by metal-on-metal and ceramic-on-metal implants from hip simulations upon the viability of L929 cells and to determine their genotoxic potential when cultured with primary human fibroblasts.

Methods: Particles were generated in a 10 station Prosim hip simulator run with water as lubricant under microseparation and standard conditions. Bearings comprised medical grade HIPed ‘BIOLOX Forte’ alumina ceramic femoral heads against Ultima metal CoCr acetabular cups (CoM) and wrought CoCr alloy ASTM F1537 femoral heads and acetabular cups (MoM). Particles were sterilised at 1800C for 4 hours and cultured with L929 fibroblasts at particle volume(μm3):cell number ratios of 500:1, 100:1, 50:1, 5:1, 0.5:1, 0.05:1, 0.005:1 and 0.0005:1. Camptothecin (1 and 2μg.ml-1) and latex beads (100μm3 per cell) were used as positive and negative controls. Cultures were for 0, 1, 2, 3, 4 and 5 days at 37oC in 5%(v/v) CO2 in air. Cell viability was assessed using the ATPlite assay. Sterile particles were cultured with primary human fibroblasts at particle volume (μm3):cell number ratios of 50:1, 5:1 and 0.5:1. Cells were exposed to 30%(v/v) H2O2 (positive control) and latex beads (50μm3 per cell; negative control). Cells were cultured for 24 hours and 5 days at 37oC in 5%(v/v) CO2 in air. Genotoxicity was assessed using the comet assay. Statistical analysis between the cell-only negative controls and the cells with the particles at various concentrations, were determined by ANOVA and calculating the minimum significant difference (MSD;p< 0.05) using the T-method.

Results: Particle volume(μm3):cell ratios of 500:1, 100:1 and 50:1 caused a significant decrease in cell viability over 5 days. Wear particles from MoM implants under microseparation wear conditions were also significantly reduced viability at particle volume(μm3):cell ratios of 5:1 over 5 days. Particles from MoM implants under standard wear conditions and CoM implants under both wear conditions resulted in increases in tail length and tail moment relative to the cells only negative control for all treatment groups after 24 hours. These decreased by day 5. Tail length and tail moment were increased at 24 hours relative to day 5 for each of the three particle types. Particles generated by MoM implants under microseparation conditions had different effects upon cells. Tail lengths increased between days 1and 5 for all particle concentrations. A significant increase in tail moments between days 1 and 5 was recorded.

Discussion: This study has shown that metal particles can cause cytotoxic effects and immediate DNA damage to fibroblasts in vitro. Particles were found to reduce cell viability over 5 days and this may account for the decreases in tail length and moments between 1 and 5 days for three particle types. This is of concern as MoM and CoM implants are designed to be implanted into young patients and, despite their low wear rates generate circa 10¹³ particles per mm3 of wear.

Introduction: Joint replacement surgery is one of the most common operations that take place in United Kingdom. The major problem in total hip arthroplasty is the generation of particulate wear debris and the subsequent biological responses. Wear debris induces osteolysis and a subsequent failure of the implant that lead to the liberation of greater quantities of particulate and soluble debris to bone marrow, blood, lymph nodes, liver and spleen. Recently, it has been suggested that these adverse effects depend not only on the chemical composition but also on the particulate nature of the material (size and shape). Particle size has been shown to influence the inflammatory response of macrophages to wear debris. This study evaluated whether particle size also influences the viability and mutagenic damage.

Methods: Cobalt chrome alloy particles of two sizes (large 2.9±1.1μm, small 0.07±0.04 μm) were generated and characterised by Scanning Electron Microscopy. Different concentrations of particles were added to primary human fibroblasts in tissue culture. The release of cytokines in the medium was assayed by Enzyme-Linked ImunnoSorbent Assay (ELISA). Cell viability was determined by MTT conversion and the degree of DNA damage was quantitatively analysed by the Alkaline Single Cell Gel Electrophoresis (COMET) assay with image analysis.

Results: Small particles initialise DNA damage at much lower volumetric concentrations (0.05 and 0.5 μm³/cell) than larger particles (500 μm³/cell). The difference in the doses was approximately related to the difference in surface area of the particles. DNA damage was related to a delayed decrease in cell viability, which was noted after three days of exposure.

In contrast, the release of the inflammatory cytokine TNF-α and the multifunctional growth factor TGF-β-2 occurred at lower doses (0.0005 to 5 μm³/cell for TNF-α and 0.5 to 50 μm³/cell for TGF-β-2). No release of IL-6 was detected at any dose. Only growth factor FGF-23 was increased in similar pattern to the DNA damage.

Conclusions: This study has demonstrated important differences between the mutagenicity, toxicity and inflammatory potential of small (nanometre sized) and large (micrometer sized) chrome particles.

Joint replacement failure is usually caused by the formation of wear debris resulting in aseptic loosening. Particulate metal and soluble metal ions from orthopaedic alloys (cobalt chromium or vanadium titanium aluminium) that are used in medical prostheses can accumulate in tissues and blood leading to increased chromosome aberrations in bone marrow and peripheral blood lymphocytes. This paper demonstrates that two of the metals used in orthopaedic prostheses, chromium and vanadium can produce delayed as well as immediate effects on the chromosomes of human fibroblasts in vitro. Fibroblasts were exposed to metal ions for only 24 hours and were then expanded over 30 population doublings involving ten passages. The initial increase of chromosomal aberrations, micronuclei formation and cell loss due to lethal mutations persisted over multiple population doublings, thereby demonstrating genomic instability. Differences were seen in the reactions of normal human fibroblasts and those infected with a retrovirus carrying the cDNA encoding hTERT that rendered the normal human fibroblasts telomerase-positive and replicatively immortal. This suggests that chromosomal instability caused by metal ions is influenced by telomere length or telomerase activity. Formerly this syndrome of genomic instability has been demonstrated in two forms following irradiation. One type is non-clonal and involves the appearance of lethal aberrations that cannot have been carried by the surviving cells. The other type is clonal and the aberrations are not lethal. These may arise as a result of complex rearrangements occurring at a high rate post-insult in surviving cells. The consequences of genomic instability are not yet known but it is possible that the increase of chromosomal aberrations that have been previously observed in human patients could be due to immediate and delayed expression of cellular damage after exposure to orthopaedic metals.

Wear debris from worn cobalt chrome joint replacements causes an increase in chromosomal translocations and aneuploidy. In this study the relationship between the amount of DNA damage and the changes in gene expression was investigated in human fibroblasts after exposure to artificial cobalt chrome particles. The comparison was made with different doses of particles, at different time intervals and in fibroblasts of different ages, those that had completed 10 population doublings (10 PD fibroblasts) and those that had completed 35 population doublings (35 PD fibroblasts). The genes (TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase 10, PURA, FRA-1 and VNR) were chosen after a previous screen with cDNA microarrays. The percentage of senescent cells was evaluated using an immunohistochemical assay for ¥â-galactosidase activity. The 35 PD fibroblasts showed significantly more ¥â-galactosidase activity than the 10 PD fibroblasts. The level of DNA damage, as detected with the alkaline comet assay, was greater at higher doses, at longer exposures (up to 24 hours) and in 10 PD fibroblasts. The expression of all the genes listed above was generally lower after exposure to cobalt chrome particles using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The reduction in gene expression, like the increase in DNA damage was greater at higher doses and at longer exposure times. After 24hr exposure the reduction in gene expression was greater in 10 PD fibroblasts compared to 35 PD fibroblasts. After 6hr exposure this was only true at higher doses of particles and the opposite was seen after a lower dose of particles. These results show that levels of gene expression of TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase10, PURA, FRA-1 and VNR may be correlated with the level of DNA damage and that this depends on the dose and length of exposure and the age of the cells. This highlights the potential importance of these genes in the mutagenicity of cobalt chrome particles in human fibroblasts.

Metal-on-metal joint replacements have been reintroduced despite some concerns regarding the potential risks posed by soluble metallic by-products. We have investigated whether there are metal selective differences between the levels of genetic damage caused to a human cell line when cultured with synovial fluids retrieved from orthopaedic joint replacement prostheses at the time of revision arthroplasty.

Methods: Synovial fluids were retrieved from revision hip and knee arthroplasty patients with bearings made from Cobalt chrome-on-Cobalt chrome, Cobalt chrome-on-polyethylene and Stainless Steel-on-polyethylene. Control synovial fluids were retrieved from primary arthroplasty cases with osteoarthritis and no implant in situ. Synovial fluid was cultured with human primary fibroblasts for 48 hours in a cell culture system under standardised conditions. The ‘Comet’ assay was used with an image analysis system to measure levels of DNA damage caused by the various synovial fluid samples. Metal levels were measured in the synovial fluid samples using atomic absorption spectroscopy.

Results: Synovial fluids from Cobalt Chrome-on-Cobalt Chrome and Cobalt Chrome-on-polyethylene joint replacements both caused substantial levels of genetic damage as detected by the Comet assay. Synovial fluids retrieved from Stainless Steel-on-polyethylene joints caused low levels of damage. The difference between these groups was highly statistically significant (p< 0.001). Control synovial fluids from osteoarthritic joints caused minimal changes. Atomic absorption spectroscopy demonstrated that the metal-on-metal synovial fluids contained substantially more cobalt and chromium than the fluids retrieved from cobalt chrome-on-polyethylene joints. Stainless steel-on-polyethylene synovial fluids contained the least metal.

Conclusions: Different alloys used in Orthopaedic implants are associated with different levels of DNA damage to cultured human cells in vitro. We are able to demonstrate that this damage is attributable at least in part to the metal content of the synovial fluid samples. We have no evidence for any long-term health risk to patients with such implants. Further research is needed in this field.

Aims: Limb sparing surgery, for selected cases of long bone primary malignancy, may be accomplished with the use of large prostheses. Conventional joint implants are known to release metal ions by corrosion or wear. The aim of this study was to determine if a specialist group of patients had elevated serum metal levels. Methods: Over a 12 month period, 20 patients who had undergone previous surgery were recruited from a bone tumour clinic. A 10ml venous blood sample was obtained and analysed for trace metals using a previously published mass-spectrometry technique. Results: Eight children (mean age 14.5 years) and 12 adults (mean age 46.5 years) were recruited a mean of 54 months and 86 months following surgery, respectively. Trace metal (aluminium, titanium, cobalt) elevation was observed in 5/8 (63%) paediatric cases and 6/12 (50%) adult cases. Three of the adults had signiþcantly raised levels, (≤ 50 times), and had undergone revision surgery for loosening. There was no observed implant loosening in the paediatric group. Conclusions: This small sample has demonstrated that many patients with long-term large tumour implants have trace metal levels below laboratory detection. Signiþcant elevation of metal levels in adults was associated with loosening or wear of implants. A signiþcant proportion of paediatric cases had slight elevations, but the signiþcance of this is unknown at present.

Aims: To study the levels of genetic damage caused to a cultured human cell line when cultured with synovial ßuid retrieved from revision arthroplasty joints. Methods: Synovial ßuids were retrieved from revision hip and knee arthroplasty patients with bearings made from Cobalt chrome-on-Cobalt chrome, Cobalt chrome-on-polyethylene, Stainless Steel-on-polyethylene and Titanium-onpolyethylene. Control synovial ßuid was retrieved from primary arthroplasty cases. Synovial ßuid was cultured with human primary þbroblasts for 48 hours in a cell culture system under standardised conditions. The ÔCometñ assay was used with an image analysis system to measure levels of DNA damage caused by the various synovial ßu id samples. Results: Synovial ßuids from Cobalt Chrome-on-Cobalt Chrome and Cobalt Chrome-on-polyethylene joint replacements caused signiþcantly (p< 0.05) more genetic damage than synovial ßuids from Stainless Steel-on-polyethylene and Titanium-on-polyethylene cases. Control synovial ßuid caused minimal change. Conclusions: Different alloys used in Orthopaedic implants are associated with different levels of DNA damage to human cells in vitro. We have no evidence for any long-term health risk to patients with such implants. Further research is needed in this þeld.

In a post-mortem study, we compared subjects with metal implants with and without visible wear with an age-matched control group to determine the extent and effects of dissemination of wear debris. In subjects with stainless-steel and cobalt-chrome prostheses metal was found in local and distant lymph nodes, bone marrow, liver and spleen. The levels were highest in subjects with loose, worn joint prostheses and the main source of the debris was the matt coating. Metal levels were also raised in subjects with implants without visible wear and, to a less extent, in those with dynamic hip screws. Necrosis of lymph nodes was seen in those cases with the most wear, and potential damage to more distant organs such as the bone marrow, liver and spleen in the long term cannot be discounted. The consequences for the immune system and the role of metal dissemination in the possible induction of neoplasia are discussed.

The production of particulate wear debris is a recognised complication of joint arthroplasty, but interest has concentrated on local tissue reactions and a possible association with implant loosening. The fate of wear products in the body remains unknown, although some of the metals used in the construction of orthopaedic implants are known to have toxic and oncogenic properties. We report histological and electron-microscopic evidence from two cases which shows that metallic debris can be identified in the lymphoreticular tissues of the body distant from the hip some years after joint replacement. The increase in the use of total arthroplasty in younger patients, the development of new alloys and the use of porous coatings must raise concern for the long-term effects of the accumulation of wear debris in the body.